Hemostatic microspheres

ABSTRACT

Provided herein are hemostatic compositions. In one embodiment, the hemostatic composition includes cross-linked polymer microspheres, such as cross-linked gelatin microspheres with pores. In another embodiment, the hemostatic composition comprises an additive such as a wetting agent, a suspending agent, or both. The hemostatic compositions may also include a hemostatic agent such as thrombin, and may include a high concentration of thrombin. The hemostatic compositions may also include plasma. Also provided herein are devices for dispersing said hemostatic compositions in a diluent, and delivering said dispersed hemostatic composition. The hemostatic compositions may also fabricated with a selected geometry as administration suggests.

RELATED APPLICATIONS

This application claims the benefit of U.S. Patent application No.61/042,156, filed Apr. 3, 2008; and U.S. Patent application No.61/150,466, filed Feb. 6, 2009, all of which are herein incorporated byreference.

FIELD OF THE INVENTION

This invention relates to hemostatic compositions, such as cross-linkedpolymers including porous cross-linked gelatin microspheres, that mayinclude hemostatic agents such as thrombin and/or plasma. In certainembodiments, the hemostatic compositions may include doses of thrombinthat encompass a range of thrombin concentrations in order to providefor rapid and reliable onset of hemostasis. In particular embodiments,the hemostatic compositions may comprise high doses of thrombin, e.g.,1000 IU/ml or higher, to provide for rapid and reliable onset ofhemostasis.

BACKGROUND OF THE INVENTION

Bleeding as a result of surgery or injury may be controlled by passivehemostats and/or hemostatic agents. Passive hemostats control bleedingmechanically, through pressure and absorption, and may be fragmented orotherwise mechanically disrupted powders, gauze, or sponges made fromoxidized regenerated cellulose, or cross-linked gelatin. Often, apassive hemostat is combined with an active hemostat, such as thrombin.There remains a need, however, for improved hemostatic compositions,particularly those that render superior clot formation.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B demonstrate that a formulation comprising thrombin andpolymer microspheres rehydrated into a gel improves clot strength andshortens clotting reaction time. FIG. 1A is a plot of clot strength withincreasing concentrations of microsphere gel. FIG. 1B is a plot of clottime (minutes reaction time) with increasing concentrations ofmicrosphere gel. ♦ IU thrombin/mg dry microspheres; ▪ microspheres only;error ±1 IU/mL Thrombin.

FIG. 2 shows that high levels of thrombin are required to overcomeheparin. A partial clot often forms in the mixing pipette when highthrombin is introduced into blood. Polymer microspheres improves themixing of thrombin with blood and improves clot formation. To partiallyovercome heparin inhibition, 75 IU to 100 IU thrombin/mL is required.The formulation of microspheres and thrombin also form a morehomogeneous clot. The blood was re-calcified with 10 mM CaCl₂, with orwithout 3.4 mg microspheres/ml gel; ♦ thrombin mixed with microspheregel; ▪ thrombin mixed directly; ▴ thrombin and microsphere gel mixedwith 1 IU/mL heparin.

FIG. 3 shows that high levels of thrombin are required to overcome theeffects of heparin. A partial clot often forms in the mixing pipettewhen thrombin is introduced into blood. Thrombin in microspheres gelimproves the homogenous mixing of thrombin with blood and improves clotformation. In the presence of 1 IU/mL heparin, almost 100-times morethrombin is required for the same reaction time without heparin. Theformulation of thrombin in microsphere gel reduces clot initiation time(R) by slowing the release of thrombin. Blood was re-calcified with 10mM CaCl₂ with or without microsphere gel, and with or without heparin. ♦thrombin mixed with 1.2 mg gelatin beads; ▪ thrombin added directly toblood; ▴ microsphere gel, thrombin, and 1 IU heparin.

FIGS. 4A and 4B graphically present expanded views of non-heparin plots.When thrombin is mixed directly with blood the clot is not homogenousbecause of rapid clot formation. The addition of thrombin to the gelatinbeads prior to mixing with blood or liquid allows better mixing to occurand improves clot reaction time and clot strength. ♦ thrombin mixed with1.2 mg gelatin beads; ▪ thrombin mixed directly with blood.

FIG. 5 is a graph presenting model estimates of time to topicalhemostasis (TTH) (mean SE) versus recombinant thrombin (rThrombin) dose.

FIGS. 6A and 6B show the Effect of rThrombin Concentration on MaximumClot Strength (G_(max)) on Heparinized Rabbit Blood pre and postTreatment with Clopidogrel. Blood samples were collected from threerabbits prior to Clopidogrel treatment and 1 IU/mL Heparin was added(top, 6A). Blood samples were taken again after Clopidogrel treatmentand in vivo heparinization (bottom, 6B). Each point represents the peakstrength G_(max) of a single TEG assay at one thrombin concentration.Thrombin concentrations ranged from 25 IU to 200 IU per mL of blood.Fitting the data to log a Dose equation demonstrates that higherthrombin concentrations are required to overcome heparin (EC50=54 IU/mL)and heparin plus clopidogrel (EC50=66 IU/mL).

FIGS. 7A-7C are graphs showing time to topical hemostasis. FIG. 7A showsdata from a rat theminephrectomy model, showing TTH of rThrombin orplacebo applied with gelatin matrix. FIG. 7B reflects data from a Rabbitliver injury model indicating TTH of rThrombin or placebo applied withgelatin matrix. FIG. 7C shows data from an A-V shunt graft puncturemodel, with TTH of rThrombin or placebo administered with gelatin matrixor as a spray.

FIG. 8 shows the TTH in a rat heminephrectomey model, comparing acomposition of thrombin and 130-380 μm polymer microspheres with acomposition of 1000 IU/mL thrombin and a 50:50 weight ratio mixture of<130 μm:130-380 μm polymer microspheres. P=mixed with placebo, rTh=mixedwith 1000 IU/mL thrombin.

DETAILED DESCRIPTION OF THE INVENTION

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention.

As used herein and in the claims, the singular forms include the pluralreference and vice versa unless the context clearly indicates otherwise.Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed herein.

The present invention provides for hemostatic compositions comprising across-linked, polymer microsphere. The hemostatic composition may be across-linked gelatin microsphere; but collagen, dextran, chitosan,alginate, protein, polysaccharide, polyacrylamide, and other hydrogelcompositions may also be used. In a particular aspect, the cross-linkedgelatin microspheres may have a diameter from about 50 μm to about 500μm. In addition, the cross-linked gelatin microspheres may furthercomprise pores having a pore diameter of about 20 μm. In certainembodiments of the invention, both the microsphere particle size and thepore diameter is optimized to maximize the desired uptake into themicrosphere and the release of the hemostat, or sustained application ofthe hemostat in the hemostatic composition in various bleedingapplications. A decrease or increase in particle size or in porediameter may enable the slow or rapid release of hemostat in thehemostatic composition depending on the application. For example,particle sizes may range from about 10 μm to about 500 μm, inclusive, intypical cross-linked gelatin microsphere. It is understood that withinthe ranges of particle sizes in the hemostatic microspheres of thepresent invention that narrower ranges within the 10 to 500 μm can beachieved, such as about 10 to 100 μm, 100 to 200 μm, 100 to 300 μm, 100to 400 μm, 200 to 300 μm, 300 to 400 μm, 400 to 500 μm, 50 to 150 μm,150 to 250 μm, 150 to 350 μm, 250 to 350 μm, 350 to 450 μm, eachinclusive, and similar incremental ranges between 10 μm and 500 μm.Moreover, smaller pore diameters from 1 μm to 50 μm, inclusive, may beemployed, as well as larger pore diameters from 50 to 200 μm, or up to300 μm, inclusive, in some applications where larger pore diameters aredesired. It is understood that within the ranges of pore diameters inthe hemostatic microspheres of the present invention that narrowerranges within the 1 μm to 300 μm, inclusive, can be achieved, such as 1to 50 μm, 50 to 100 μm, 100 to 150 μm, 150 to 200 μm, 200 to 250 μm, 250to 300 μm, 10 to 60 μm, 20 μm to 70 μm, 30 to 80 μm, 40 to 90 μm, 60 to110 μm, 70 to 120 μm, 80 to 130 μm, 90 to 140 μm, each inclusive, andsimilar incremental ranges up to about 300 μm.

Haemostatic agents such as thrombin and/or plasma may be used. Incertain embodiments, the hemostatic compositions may comprise thrombin.Such thrombin may be animal- or human-plasma derived, or may berecombinant thrombin such as recombinant human thrombin (rThrombin).Moreover, additional hemostatic agents may be used in addition tothrombin, such as fibrinogen, factor XIII, Protein C, epinephrine,thrombomodulin, factor V, factor VIII, and the like.

Further to this aspect, the hemostatic composition may be mixed with awetting agent, for example poloxamer or poloxamer 188, polyethyleneglycol, or polysorbate. Alternatively, the hemostatic composition may bemixed with a suspending agent such as carboxymethyl-cellulose. Thehemostatic composition with or without wetting agent and/or suspendingagent may be prepared as a dry powder, or as pre-formed geometries wherethe hemostatic composition is compressed, dried, chemically bound, orthermally formed into a desired configuration. In a further aspect ofthis embodiment, an active hemostat, e.g., thrombin, is combined withthe diluent used to disperse said cross-linked microsphere prior toadministration. In a particular embodiment, the diluent comprisesplasma, which may be derived from a patient's own blood. In a furtherembodiment, the microspheres are suspended in a diluent of sufficientviscosity, adhesiveness and density that application in a non-gravitydependent manner may occur.

Another embodiment provides for a hemostatic composition comprising across-linked polymer (e.g., cross-linked gelatin) microspheres and atleast one additive. In one aspect, the additive is a wetting agentand/or a suspending agent. The additive may be a wetting agent, such aspoloxamer or poloxamer 188, polyethylene glycol, or polysorbate.Alternatively, the additive may be suspending agent, such ascarboxymethylcellulose. The hemostatic composition may be a dry powder.In a further aspect of this embodiment, an active hemostat, e.g.,thrombin, is combined with the diluent used to disperse saidcross-linked polymer microsphere and one or more additives. The diluentmay comprise plasma, such as plasma prepared from the subject receivingthe hemostatic composition.

The hemostatic composition mixed with additive can comprise a pluralityof porous, cross-linked microspheres. The cross-linked gelatinmicrospheres may be mixed with a wetting agent, such as poloxamer 188,in a weight-to-weight ratio ranging from 60:1 to 3:1 (ratio of gelatinmicrosphere:poloxamer 188), inclusive. To prepare for application to atarget site, the hemostatic composition is easily and substantiallyhomogenously dispersed in an aqueous vehicle, yielding the consistencyof a fully-hydrated paste or gel. The hemostatic compositions of thepresent invention are prepared at the point of use, yet they maintainphysical properties to provide syringeability and flowability overextended time periods (e.g., hours).

The present invention also includes methods of making and sterilizingmedical devices containing the hemostatic compositions disposed therein.

Another embodiment of the present invention provides for a hemostaticcomposition carrying a range of thrombin doses from low to high doses ofthrombin. More specifically, porous microspheres may be charged with 125IU to 700 IU thrombin, inclusive; 700 IU to 1,000 IU, inclusive; 1,000IU; 2,000 IU; or up to 5,000 IU thrombin per mL rehydrated microspheregel, inclusive. In certain applications porous microspheres may becharged with over 1,000 IU thrombin per mL rehydrated microsphere gel,over 2,000 IU thrombin per mL rehydrated microsphere gel, for example upto 5,000 IU per mL and up to 50,000 IU per mL rehydrated microspheregel, inclusive. These compositions release a high level of thrombin,yield a homogenous clot, and are especially useful compositions forapplications in blood containing a blood-thinner such as aspirin orheparin or an anti-clotting agent such as clopidogrel bisulfate (e.g.,Plavix® or other brand).

An alternative embodiment comprises a geometric hemostatic device shapedfrom a compressed hemostatic composition, that may be applied by commonsurgical instrumentation or as part of specially designedinstrumentation for use in endoscopic, microscopic and robotichemostatic applications. For example, the porous microspheres may becompressed, dried, chemically bound, or thermally formed intocylindrical shapes that retain their shape and rigidity or that achievea certain flexibility after wetting. Such shaped compositions aresuitable for introduction via an endoscope, and thus serve as areservoir to thrombin delivery, as well as a firm extension that may begrasped or attached to endoscopic instruments for the simultaneousapplication of pressure and thrombin. Alternatively, the porousmicrospheres may be compressed, dried, chemically bound or thermallyformed into shapes that conform to surgical anatomical structures thatrequire application of thrombin via a fluid (thrombin) retaining,pressure transmitting substrate. Such shapes could include annularshapes and semi-annular shapes for placement around vascular anastomosesof various sizes. They may also be shaped to conform to predictablesurgically-induced cavity defects, such as those arising from breast“lumpectomy”, or other surgically-induced cavity defects.

A further embodiment provides for a hemostatic composition in a deliverydevice for application of said hemostatic composition to a site ofinterest. In one aspect of this embodiment the delivery device is asyringe. In a further aspect of this embodiment, said syringe isattached to a delivery tip.

A further embodiment provides for a method of applying a hemostaticcomposition to a target site with the objective of reducing bleeding. Inone aspect of this embodiment, the hemostatic composition is applied toa target site (bleed) using a piece of gauze. In this aspect, thehemostatic composition is applied to the gauze and then the gauze andthe hemostatic composition are applied to a target site, thus bringingthe hemostatic composition in contact with the bleed. The method of thepresent embodiment provides for a stronger clot when compared to bloodalone.

In another aspect the hemostatic composition is applied to a target sitevia its dispensing from a syringe. In this aspect, a syringe is loadedwith a hemostatic composition and the syringe is directed towards thetarget site. The hemostatic composition is then extruded from thesyringe to the target site. Optionally, a piece of gauze is then appliedover the hemostatic composition at the target site, whereupon mechanicalpressure is applied by a surgeon. In a further aspect of thisembodiment, a syringe attached to a tip is loaded with a hemostaticcomposition and the syringe and tip are directed towards the targetsite. Hemostatic composition is then extruded from the syringe to thetarget site. Tips are useful, for example, when the target site is at alocation that is not readily accessible to a syringe alone. Such placesinclude target sites that are deep within a cavity, are partiallyobstructed by an organ, or others. Tips are also useful for controllingthe size of the extruded hemostatic composition.

For example, a tip can be useful allowing the user to extrude acontinuous line of hemostatic composition to a lengthy bleed site.Optionally, a piece of gauze is then applied over the hemostaticcomposition at these target sites.

An embodiment of the present invention provides for an efficacious,flowable hemostatic composition, which may comprise a cross-linkedgelatin microsphere that exhibits minimal but rapid and completeswelling; minimal “stickiness;” and acceptable syringeability upondispersion in aqueous solution. More specifically, the embodimentprovides for a significantly porous polymer microsphere, e.g.,cross-linked gelatin microsphere, wherein the pores increase theparticle surface area, thereby increasing contact activation at theprocoagulant surface to facilitate hemostasis. Further, the pores canentrap an active hemostat, such as thrombin, thereby increasing theretention of the active hemostat at the application site. Indeed, thehemostatic composition may carry a higher dose of thrombin than has beendescribed previously. These cross-linked gelatin microspheres are animprovement over the fragmented hydrogels currently used as passivehemostats.

Particular embodiments also provide for porous microspheres that are notadministered in powder form. Instead, these porous microsphere,hemostatic compositions are bound into geometries that replicate currentgelatin sponge conformations (rectangular, hexahedron shapes) or thatinclude any and all other potential conformations. For example, porousmicrospheres may be compressed, dried, chemically bound, or thermallyformed into cylindrical shapes that retain their shape and rigidity orthat achieve a certain flexibility after wetting and are suitable forintroduction via an endoscope, and thus serves as a reservoir forthrombin delivery as well as a firm structure that may be grasped orattached to endoscopic instruments for the simultaneous application ofpressure and thrombin. Alternatively, the porous microsphere hemostaticcomposition may be compressed, dried, chemically bound or thermallyformed into shapes of all kinds that conform to surgical anatomicalstructures that require application of thrombin via a fluid (thrombin)retaining, pressure transmitting substrate. Such shapes could includeannular shapes and semi-annular shapes for placement around vascularanastomoses of various sizes, as well as shapes that conform topredictable surgically-induced cavity defects such as those arising frombreast “lumpectomy” or other surgically-induced cavity defect.

The efficacy and usability of cross-linked gelatin powders are dependenton the fraction and degree of cross-linking, where the fraction ofcross-linked gelatin out of the total gelatin describes the fraction ofgelatin insoluble at body temperature (37° C.) and the degree (alsoreferred to as extent) of cross-linking is a measure of the amount ofcross-links within 37° C. insoluble, cross-linked gelatin. If thefraction cross-linked is too low, soluble gelatin present in thedispersed gelatin matrix reduces the concentration of suspendedparticles responsible for the hemostatic effect that can lead to reducedefficacy marked by bleed-through. Also, these preparations can havereduced efficacy because the soluble gelatin can render the dispersedproduct “stickier,” which leads to re-bleeding following removal ofgauze or other materials often used to aid in pressure application tofacilitate hemostasis. If the degree of cross-linking is too high, thecross-linked gelatin can be rendered too hydrophobic to allow for easyand homogeneous dispersion in aqueous solutions, thereby reducingusability (i.e., syringeability). If the degree of cross-linking is toolow, the cross-linked gelatin can be too absorptive leading to extendedswelling times with varied consistency over time subsequent todispersion. Excessive swelling after application of partially hydratedabsorbents can also potentially lead to serious adverse reactions suchas paralysis and nerve damage if hemostats are used in, or in proximityto foramina in bone, areas of bony confine, the spinal cord, and/or theoptic nerve and chiasm.

The degree and extent of gelatin cross-linking is also affected byionizing radiation (i.e., e-beam or γ-irradiation) often used toterminally sterilize medical devices. Although the ionizing radiationdose required for terminal sterilization is dependent on the productbioburden prior to irradiation, a typical sterilizing dose ofγ-irradiation for medical devices is 25 kGy; a 25 kGy targetγ-irradiation dose often exposes the products to a range of 15 kGy to 35kGy. The fraction cross-linked and degree of cross-linking can bereduced when cross-linked gelatin is irradiated as a dry powder. Thedegree and extent of gelatin cross-linking can be increased, however,when cross-linked gelatin is irradiated as hydrated dispersions. Assuch, the properties, such as solubility, hydrophobicity and/orswelling, of the terminally sterilized cross-linked gelatin aredependent on the degree and extent of cross-linking prior toirradiation, as well as the form that is irradiated (i.e., hydrateddispersion or dry powder). Irradiating cross-linked gelatin that has alimited fraction cross-linked and degree of cross-linking as a hydrateddispersion could lead to intra-batch variability due to the range ofirradiation exposure (e.g., 15 kGy to 35 kGy).

Ideally, a gelatin cross-linking process produces a product with asufficient degree and extent of cross-linking so that any changesinduced by exposure of a dry powder to a range of ionizing radiationdoses (e.g., 15 kGy to 35 kGy) are inconsequential with regard tosolubility, hydrophobicity, and/or swelling. Gelatin can be cross-linkedusing a dehydrothermal or chemical process that utilizes cross-linkingagents such as glutaraldehyde or hexamethylene diisocyanate.Cross-linked gelatin having low fraction of cross-linked gelatin anddegree of cross-linking have significant increases in solubility,swelling, and “stickiness” induced if irradiated as a dry powder becausethe degree and extent of cross-linking is reduced. Irradiatingcross-linked gelatins with a limited fraction and degree ofcross-linking as a hydrated paste leads to intra-batch variability insolubility, hydrophobicity, and stickiness due to product exposure overan irradiation dose range (i.e. 15 kGy to 35 kGy).

Existing commercial cross-linked hydrogel products are supplied eitheras a dry powder or a partially hydrated paste intended foradministration after dispersion in an appropriate amount of aqueousvehicle. These powders are formed by mechanical disruption ofcross-linked matricies, such as absorbable gelatin sponges, U.S.P.(e.g., GELFOAM®, Pfizer, Inc. or SURGIFOAM™, Ethicon, Inc.), or thecakes that are Rained during typical chemical or dehydrothermalcross-linking treatment (see, e.g., U.S. Pat. No. 6,063,061; U.S. Patentapplication pub. No. 2003/0064109). These cross-linked hydrogels aremost typically gelatin based hydrogels, however, collagen, dextran,chitosan and other compositions are also used, as is know to one skilledin the art.

Hydrogel-based hemostatic compositions may be administered dry,partially hydrated, or fully hydrated. In the fully hydrated state, thehydrogel can not absorb further fluid, and is fully swollen in size. Incontrast, a dry or partially hydrated hydrogel composition has excessadsorptive capacity. Upon administration, dry or partially hydratedhydrogel will absorb fluid leading to a swelling of the gelatin matrixin vivo. Excessive swelling after application can potentially lead toserious adverse reactions, such as paralysis and nerve damage ifhemostats are used in, or in proximity to, foramina in bone, areas ofbony confine, the spinal cord, and/or the optic nerve and chiasm. Hence,swelling of dry or partially hydrated hydrogel should be considered inthe context of administration.

The ideal hemostatic composition has at least one the followingproperties: it is compatible with active hemostats, such as thrombin; ithas limited aqueous solubility of the hemostatic gelatin matrix; itexhibits minimal changes in efficacy and usability after exposure to awide range of ionizing radiation (15 kGy to 35 kGy) sufficient to yielda terminally-sterilized product; it shows rapid and complete swellingwhen dispersed in aqueous vehicle; it is effective when administeredfully-hydrated; it has acceptable syringeability, allowing completedispensing of a homogeneous dispersion from a syringe (or deliverydevice) with minimal force; it contains significant porosity; it has ashort resorption time after administration (less than one year, or lessthan six months); its particle shape and size facilitates flowproperties as both dry powder and dispersed suspension (e.g., gel).

In a further embodiment, the present invention utilizes a porous,cross-linked gelatin hydrogel microsphere combined with a wetting agentand/or a suspending agent resulting in a flowable cross-linkedcomposition that exhibits minimal but rapid and complete swelling,minimal stickiness and improved syringeability upon dispersion inaqueous solution. The chemically cross-linked microsphere may bemanufactured by an emulsion process that is specifically designed toproduce approximately spherical microparticles and introduce pores of anaverage size of about 20 μm and yields a microsphere product of definedparticle size range (about 50 μm to about 500 μm). The microspheres canbe manufactured according to U.S. Pat. No. 7,404,971, U.S. Pat. No.4,935,365, U.S. Pat. No. 5,015,576. Microspheres are also availablecommercially, for example, CultiSpher®-S macroporous gelatinmicrocarrier microspheres (Celltrix, Malmo, Sweden; Percell Biolytica,Åstorp, Sweden). Hemostatic efficacy has been established as a fullyhydrated dispersion in non-clinical models.

One advantage as a hemostat of the cross-linked gelatin microspherepowder prepared by an emulsion process containing pores (versusmicrosphere powder without pores) of a defined particle size range (thatexcludes fine and course particles) has been established usingnon-clinical bleeding models. Another advantage of the cross-linkedgelatin microsphere process is to yield a dry powder that is resistantto changes over a range of γ-irradiation doses (15 kGy to 35 kGy)compared with cross-linked gelatins in the art, in terms of thoseproperties necessary for efficacy of a flowable passive hemostat (i.e.,solubility, hydrophobicity, swelling). The spherical particle shape anddefined size distribution also facilitates powder flow properties thataid manufacturing (e.g., filling of the dry powder into a deliverydevice) and dispersion in aqueous vehicles. Although the porousmicrospheres of the current invention provide the previously discussedadvantages as a hemostat, the particles have a “sponging-out” effectwherein aqueous solution is removed from a hydrated dispersion when saiddispersion is used in a delivery device that relies upon mechanicalforce for delivery of said hydrated dispersion. For example, when theporous, cross-linked microspheres are dispersed in an aqueous solutionand the delivery device is a syringe, the mechanical force applied tothe syringe plunger causes a sponging-out of the aqueous solutiondisproportionately to the porous, cross-linked microsphere. As a result,the initially dispersed material has a more dilute consistency than doesthe later dispersed material. Moreover, the later-dispersed material maybecome so dry from the sponging-out effect that this later material willnot disperse from a syringe with reasonable force.

The sponging-out effect is ameliorated, however, by inclusion of wettingagents (e.g., poloxamer 188, polyethylene glycol 3350, polysorbate 20 orpolysorbate 80) and/or suspending agents (e.g., carboxymethyl-cellulose)as additives. The wetting agent may be mixed with a porous, cross-linkedgelatin microsphere in an appropriate ratio dependant on the agent used.Regarding poloxamer-188, for example, a weight-to-weight ratio of 60-3:1(cross-linked gelatin:additive) is effective. Similarly, the suspendingagent may be mixed with a porous, cross-linked gelatin microsphere inweight-to-weight ratio of 60-3:1 (cross-linked gelatin:additive). If amixture of wetting agent and suspending agent are mixed with the porous,cross-linked gelatin microsphere, the wetting agent plus suspendingagent are mixed with a porous, cross-linked gelatin microsphere in anappropriate ratio. The suspending/wetting agents may also be introducedvia the vehicle used to disperse the microspheres, as could be done forthe polysorbates. The combination of cross-linked gelatin microspherepowder and additive ensures the desirable properties of the flowablepassive hemostat (i.e., homogeneity of dispersions, minimal extrusionforce) are retained for extended time periods (hours). The describedformulation is also compatible with thrombin.

In several embodiments of the present invention, the hemostaticcomposition includes thrombin. As used herein, “thrombin” denotes theactivated enzyme, also known as alpha-thrombin, which results from theproteolytic cleavage of prothrombin (factor II). Thrombin can beprepared by a variety of methods known in the art, and the term“thrombin” is not intended to imply a particular method of production.Both human and non-human thrombins can be used within the presentinvention. Thrombin is used medically as a hemostatic agent and as acomponent of tissue adhesives. Human and non-human (e.g., bovine)thrombins are prepared according to methods known in the art.Purification of thrombin from plasma is disclosed by, for example,Bui-Khac et al., U.S. Pat. No. 5,981,254. Purification of thrombin fromplasma fractions, such as Cohn fraction III, is disclosed by Fenton etal., 252 J. Biol. Chem. 3587-98 (1977). Recombinant thrombin can beprepared from a prethrombin precursor by activation with a snake venomactivator as disclosed in U.S. Pat. No. 5,476,777. Thus, the thrombinmay be a recombinant thrombin. The amount of the recombinant thrombin inthe formulation may be between 3000 NIH (National Institutes of Health)Units and 30,000 NIH Units of recombinant thrombin, inclusive, or 5000NIH Units of recombinant thrombin. In this aspect, the thrombin may beprovided in the kit as a lyophilized powder (see, e.g., U.S. Pat. No.7,473,543). This lyophilized powder can be reconstituted using adiluent, including a diluent comprising plasma.

In another embodiment of the invention, when the thrombin is added todry microsphere gel and then introduced into blood, the rate of clotformation is increased, indicating that the clot kinetics is slower thanwhen thrombin is added directly into blood (FIG. 4A). When combined withthe cross-linked polymer microspheres of the present invention, thrombinalso yields better clot strength (FIG. 4B). Without the microsphere gel,the clot strength declines with increasing thrombin, whereas thethrombin plus microsphere gel maintains clot strength. In this regard,thrombin may be included in the hemostatic composition of the presentinvention at a concentration of about 1000 IU/mL. This data alsosupports that higher thrombin concentrations may be used in haemostaticcompositions containing microsphere gels. Without being bound by theory,passive diffusion of the thrombin from the high-thrombin-dose hemostaticcomposition may aid in the formation of the homogenous, strong clot. Theimportance of this formulation is readily apparent in blood that hasbeen treated with a thinner or anti-clotting agent such as aspirin,heparin, or clopidogrel bisulfate. In the case of such treated blood,the high-dose thrombin microspheres hemostatic composition is able todeliver ten-times the thrombin found in normal blood and yield a normalblood clot. The importance of this formulation is also readily apparentin surgical or other bleeding applications where formation of thehomogenous, strong, clot is desirable in the absence of such thinner oranti-clotting agent.

As used herein, “matrix” denotes a mixture containing at leastmicrosopheres and a hemostatic agent. A matrix may or may not alsocontain a wetting agent. For example, as used in some examples herein, amatrix includes cross-linked gelatin microspheres, thrombin (e.g.,rThrombin) as a hemostatic agent and a poloxamer such as poloxamer 188.It is understood that a matrix may contain different mixtures ofmicrosopheres, a hemostatic agent, and one or more wetting agents asdescribed herein.

As used herein, “dispersion” denotes a mixture containing at least twophases (for example, a mixture containing a solid and a liquid phase).Depending on the viscosity of a dispersion, it may be considered asuspension or a paste. Microspheres in the hemostatic compositions ofthe present invention can be dispersed in an aqueous vehicle, includingan aqueous vehicle comprising plasma.

Since the 1940s, thrombin has been used during surgical procedures as atopical hemostatic agent to speed time to hemostasis (TTH) and improvevisualization of the surgical field, and for use in procedures such asincluding use in burn patients undergoing debridement and skin grafting(Bishop et al., 32(S1) Semin. Thromb. Hemost. 86-97 (2006); Lundblad etal., 91(5) Thromb. Hemost. 851-60 (2004)). The safety and efficacy of1000 IU/mL of topical thrombin was recently confirmed in human clinicaltrials (Chapman et al., 205(2) J. Am. Coll. Surg. 256-65 (2007); Doriaet al., 24(3) Curr. Med. Res. Opin. 785-94 (2008)), but human clinicaltrials have not compared the effects of differing concentrations oftopical thrombin on hemostatic efficacy. The critical nature of thrombinconcentration in fibrin clot formation has been demonstrated in a numberof in vitro settings, however, indicating that clots formed in thepresence of high concentrations of thrombin have more tightly packedfibrin strands.

Indirectly, variations in time to hemostasis in vivo may indicaterelative effects of topical thrombin concentration on clot integrity. Arecent evaluation in a porcine liver injury model of human thrombin plusgelatin sponge at 125 IU/mL showed improved activity over saline plusgelatin sponge (Adams et al., J. Thromb. Thrombolysis [0929-5305] (Jul.16, 2008)). Even with a liberalized definition of hemostasis (limitedoozing was also a permitted endpoint), limited accumulative hemostasiswas observed after the first 3-minute time point. There were also asignificant number of sites rebleeding during the 12-minute evaluationperiod, however, pointing strongly to concentration limited hemostaticeffect. Those observations raise the question of whether there is adifference in onset of hemostasis and clot integrity between thestandard 1000 IU/mL and the lower 125 IU/mL application (Adams et al.,2008).

Based on these observations, the adoption of 1000 IU/mL thrombinconcentration may have evolved in clinical practice because of observedefficacy in a range of clinical settings that included both pathologicand pharmacologic clotting derangements. It stands to reason that thepotency of the thrombin enzyme in coagulation would enable lowerconcentration of topical thrombin to be effective in some, but not allclinical settings. For example, when high thrombin is added directlyinto blood by standard pipetting techniques the fibrinogen is convertedto fibrin faster than mixing can occur, which results in anon-homogenous clot. Hence, to test the effect of thrombin concentrationon time to hemostasis (TTH) under varying conditions of pharmacologicanticoagulation and platelet impairment, a range of thrombinconcentrations were evaluated in a model of brisk arterial anastomoticbleeding in rabbits. Parallel evaluations of clot viscoelasticproperties were performed by modified thromboelastography. To examinewhether clot integrity was of potential clinical significance, clotburst at the site of bleeding was evaluated.

Thrombin concentration during fibrin clot formation determines clotintegrity at the time of hemostasis. Numerous factors work to reduceboth endogenous and exogenous thrombin concentration at the bleedingwound interface: removal and dilution by hemorrhagic blood flow, rapidbinding to inhibitors such as ATIII, entrapment in developing thrombus,and mechanical removal by sponge and/or irrigation. Whether topicalthrombin is applied or not, surgeons rely on intraoperative grossevaluations of hemostasis as predictors of whether hemostasis will bedurable after wound closure. As a practical matter, the consequences ofinadequate clot structure are observed when rebleeding or hematomaformation occurs. When hemostasis is delayed, coagulability is usuallyassessed by PT, PTT, and platelet count with or without plateletfunction measurements. In addition, thromboelastography (TEG), an exvivo analysis of time dependent viscoelastic changes during clotformation may be performed as a means for rapidly detecting pathologicderangements in clotting. These laboratory assessments may not directlycorrelate with intraoperative bleeding severity nor do they predictresponse to surgical intervention due to the many variables influencingclot formation in the wound. Not all of these variables are understoodin real time, with the predictable result that current depictions of thefibrin polymerization and platelet incorporation during clot formationare highly stylized.

Multiple in vitro experiments have indicated that thrombin concentrationis the most critical factor during fibrin clot formation, and clotsformed in the presence of high concentrations of thrombin have moretightly packed fibrin strands (Blomback et al., 997 (1-2) Biocim.Biophys. Acta 96-110 (1989); Blomback et al., 75(5) Thromb. Res. 521-38(1994); Wolberg, 21(3) Blood Rev. 131-142 (2007)). In addition, normalplatelet function is required for physiologic clot initiation. One grouphas described the thrombin concentration dependency of hemostasis in aseries of controlled in vivo bleeding models. Exogenous rThrombin waseffective in achieving hemostasis in an in vivo rabbit model for hepaticbleeding related to surgery, at doses between 500 IU/mL and 2000 IU/mL(Heffernan et al., 47(1) Regul. Toxicol. Pharmacol. 48-58 (2007)). Inthis model, rThrombin was effective in stopping bleeding in adose-dependent manner when applied with gauze pads. A similar rabbit invivo model used rThrombin at concentrations from 100 IU/mL to 2000 IU/mLwith either gauze sponges or absorbable gelatin sponges, and also showedreduced time to hemostasis (TTH) which was dependent on the rThrombindose (Meehan & Bolton, 121(2) J. Surg. Res. 323 (2004)).

The rabbit in vivo model presented herein replicates vascularanastomotic bleeding with a rabbit arterial venous (AV) grafts model.This model was used to evaluate 31.25 IU/mL, 62.5 IU/mL, 125 IU/mL, and1000 IU/mL rThrombin in combination with absorbable gelatin sponge, USP,and the effect on TTH. In other assays, TTH was evaluated using twoconcentrations of rThrombin, 125 IU/mL and 1000 IU/mL, as hemostaticagents in combination with an absorbable gelatin sponge, USP, in rabbitsthat had been pretreated with clopidogrel bisulfate, heparin, or both.In animals treated with heparin only, both rThrombin concentrationsaccelerated hemostasis. Notably, the standard error around the TTHachieved by 125 IU/mL was much broader than that of the 1000 IU/mLtreated animals. In clopidogrel bisulfate-treated animals, rThrombin at1000 IU/mL achieved hemostasis at the same time point as in theheparin-only-treated animals. In contrast, clopidogrel bisulfateinhibition of platelet function was not overcome by the application of125 IU/mL rThrombin. The reason for this disparity in efficacy is notintuitively obvious, and concentration-dependent thrombin reversal ofthe effects platelet inhibition on coagulability has not been reported.This lends credence to the widespread use of topical thrombinpreparation in vascular and cardiac surgery; because thrombin at 1000IU/mL speeds hemostasis over the full spectrum of clinical bleedingchallenges.

An active test of clot integrity was performed by clamping the graftfollowing the achievement of hemostasis (clot burst testing). Theresults suggest that there is another effect of thrombin concentrationthat needs to be considered: clot stability. This provocative testmeasures the adhesiveness of the clot boundary to the PTFE graftmaterial as well as platelet force development. Platelet forcedevelopment is a process of thrombus maturation in which plateletscontract with the consequence of increasing fibrin strand density thusensuring that a sudden spike in pressure does not lead to clot failure.Pharmacologic and mechanical inhibition of platelet function has beenassociated with increased bleeding in humans after cardiopulmonarybypass (Greilich et al., 105(6) Thromb. Res. 523-29 (2002)). In thislight, it is likely that the rebleeding wounds evaluated in the porcinehepatic bleeding model at 12 minutes rebled secondary to reduced clotstrength and density. Notably, there was no reasonable means by whichclot burst could have been studied in that model, because hepaticbleeding is generally low pressure, venous bleeding.

Additionally, a thromboelastograph technique (TEG) was used in vitro toexamine clot strength using samples in vitro from rabbits in the in vivoAV shunt experiments. Blood clots have both viscous and elasticproperties, and the thromboelastograph has been used to measure the clotstrength (elastic shear modulus) of clotting blood, and has beendemonstrated to measure elastic properties independent of viscosity(Chandler, 21(S4) Seminars Thromb. & Hemost. 1-6 (1995)). Becauseexogenous thrombin causes almost immediate clotting, it was necessary toalter the conditions typically used for TEG experiments so that thereaction could be slowed. The in vitro TEG experiments were also used todemonstrate the effect various anticoagulants had on clot strength, andthe interaction of various concentrations of rThrombin whenanticoagulants were present. Although the TEG experiments requirerThrombin concentrations that cannot be compared directly with theconcentrations of rThrombin used in the rabbit AV shunt model, the dataconfirm the concentration dependent reversal of the effects ofclopidogrel bisulfate platelet dysfunction on clot formation.

The present work evaluates the effects of thrombin concentration onthree areas of clinical hemostasis pertinent to every surgical practice:time to cessation of bleeding across a range of pharmacologicalcoagulation inhibition, clot strength and resistance to clot disruption.The observation that rThrombin at 1000 IU/mL negates the effects ofclopidogrel bisulfate on time to hemostasis has significant implicationsfor clinicians. Clopidogrel bisulfate irreversibly inhibits ADPreceptors on platelets and there is a wide variety of opinion regardingthe timing of discontinuation clopidogrel bisulfate prior to surgery. Inpatients with high risk of perioperative MI, it has been argued thatstopping clopidogrel bisulfate may affect the incidence of adversecardiac events. Thus, an ever increasing percentage of patients arecoming for urgent, emergent and elective procedures with significantclopidogrel bisulfate platelet inhibition. The ability of 1000 IU/mLtopical rThrombin to negate the impact of clopidogrel bisulfate plateletinhibition on time to hemostasis may mitigate the bleeding risks thatresult from those changes in surgical population.

Additionally, surgical treatment of coagulation impaired patients hasevolved over time. In this study, the thromboelastographic confirmationof the thrombin concentration dependence on clot strength furthersupports the idea that fibrin clot density is a function of availablethrombin concentration. Consequently, durable hemostasis was in alllikelihood a key driver for the evolution of thrombin 1000 IU/mL as thestandard concentration for most surgical applications.

Moreover, the observation that clots formed in the presence of thehigher thrombin concentration were more resistant to clot disruption isimportant as evidence of the differences in clot structure andmaturation that occur at differing thrombin concentrations. Persistenthemostasis that resists the stresses of the early recovery period is adesirable outcome for all surgery. Thus, the use of topical rThrombin at1000 IU/mL is reasonable as a standard of care. Although both 125 IU/mLor 1000 IU/mL rThrombin will shorten the time to onset of hemostasiswhen applied with absorbable gelatin sponge, the superior clot structureand maturation occurring at the higher thrombin concentrations suggestthat higher concentrations of thrombin may perform better in a clinicalsetting. rThrombin will consistently shorten time to hemostasis over arange of clinical conditions that mimic the current surgical population,however, although the current use of thrombin 1000 IU/mL is the standardof care for topical hemostasis, use of higher concentrations of thrombinin such applications may be justified.

More specifically, as detailed in the Examples below, a modified,heparinized rabbit arterio-venous (AV) shunt preparation was selected tomodel vascular anastomotic bleeding. Standardized,polytetrafluoroethylene (PTFE) arterial venous grafts were puncturedwith a suture needle, immediately wrapped with a thrombin or placebocontaining absorbable gelatin sponge, USP, and covered by gauze spongesapplied with continual pressure. Bemostasis was assessed using astandardized procedure at regular intervals. In the first set ofexperiments (heparin only), an absorbable gelatin sponge, USP, wasrandomly combined with saline, 31.25 IU/mL, 62.5 IU/mL, 125 IU/mL, or1000 IU/mL of rThrombin, and time to hemostasis (TTH) was assessed by ablinded observer. In a similar second set of experiments (heparin plusclopidogrel bisulfate), AV shunts were inserted and treatment wasrandomized to placebo, 125 IU/mL or 1000 IU/mL rThrombin, in combinationwith the absorbable gelatin sponge, USP followed by blinded TTHassessment. In preparations that achieved hemostasis, binary clot burstchallenges were performed at 5 minutes by rapid clamping of the distalAV graft. Determination of rThrombin concentration effect on clotviscoelastic strength was obtained by serial evaluations of ex-vivosamples using thromboelastographic (TEG) methods.

In the rabbit AV shunt model, increasing concentrations of rThrombindecreased TTH in a dose dependent manner. When rabbits were pretreatedwith clopidogrel bisulfate, TTH was significantly lower when 1000 IU/ml,of rThrombin was used in conjunction with an absorbable gelatin sponge,as compared to 125 IU/mL of rThrombin. Furthermore, TTH in the presenceof 1000 IU/mL rThrombin was highly reproducible, while TTH at the lowerconcentration varied widely. The clots formed by the 1000 IU/mL ofrThrombin were also less likely to rupture during the clot burstassessment than those formed in the presence of 125 IU/mL of rThrombin.In addition, TEG measurements demonstrated that the rate of clotformation and the strength of clots formed in vitro were dependent onthe concentration of rThrombin, particularly in the presence ofanticoagulants such as clopidogrel bisulfate.

Thus, in an animal model designed to mimic clinical coagulationdysfunction, topical rThrombin 1000 IU/mL provided rapid, reliable onsetof hemostasis when compared to rThrombin 125 IU/mL. The paradigm thatthrombin concentration is the key determinant of time to onset ofhemostasis and clot strength holds true even in the presence ofsignificant heparinization and potent platelet inhibition.

In another embodiment, the hemostatic composition is provided in a kit,wherein said kit further comprises one or more of; a first syringe, asecond syringe, a syringe tip, a diluent, an additive, and thrombin.Optionally, the kit also includes materials suitable for the use ofpatient plasma in or as the diluent. In an aspect of this embodiment,the hemostatic composition is present within the barrel of said firstsyringe. The hemostatic composition may comprise a cross-linked gelatinmicrospheres and is present within the barrel of said syringe as a drypowder. Alternatively, the hemostatic composition is cross-linkedgelatin microspheres and is present within the barrel of said syringe aspartially or fully hydrated paste or gel. In a further aspect of thisembodiment, the hemostatic composition is mixed with an additive and ispresent within the barrel of the first syringe. Some of the hemostaticcomposition may be cross-linked gelatin microspheres mixed with anadditive, and is present within the barrel of the syringe as a drypowder. Alternatively, the hemostatic composition is a cross-linkedgelatin microspheres and is mixed with as additive are present withinthe barrel of said syringe as partially or fully hydrated paste or gel.As is used herein, powdered hemostatic compositions having a moisturecontent below 20% by weight are considered dry powders.

In another aspect of this embodiment, there is provided a hemostaticcomposition mixed with an additive. The hemostatic composition may be across-linked gelatin microsphere. In this aspect, said cross-linkedgelatin microspheres have a diameter from about 50 μm to about 500 μm.In addition, the cross-linked gelatin microspheres may further comprisepores, and the pores may have a pore diameter of about 20 μm. Further tothis aspect, the hemostatic composition is optionally mixed with anadditive that is a wetting agent such as poloxamer or poloxamer 188,polyethylene glycol, or polysorbate. Alternatively, the hemostaticcomposition is mixed with an additive that is a suspending agent such ascarboxymethylcellulose. The hemostatic composition with or withoutwetting agent and/or suspending agent may be a dry powder.

In a further aspect of this embodiment provides for a hemostaticcomposition mixed with an additive wherein said hemostatic compositionis a cross-linked polymer and said additive selected from the groupconsisting of a wetting agent, a suspending agent, and both a wettingagent and a suspending agent, cross-linked polymer is gelatin; howevercollagen, dextran, chitosan, alginate and other compositions may also beused. The gelatin may be dehydrothermally cross-linked, or chemicallycross-linked, or cross-linked via other means such as irradiation. Thecross-linked polymer can be in any shape, such as a cross-linked gelatinmicrosphere, a cross-linked gelatin microsphere further comprising poreshaving a diameter from about about 50 μm to about 500 μm, inclusive, across-linked gelatin microsphere having a diameter from about about 50μm to about 500 μm, inclusive, and further comprising pores and saidpores having a pore diameter of about 20 μm. The hemostatic compositionmay be formulated into a dry powder. The additive may be a wetting agentsuch as poloxamer or poloxamer 188. Alternatively, the additive may be asuspending agent such as carboxymethylcellulose. Cross-linked polymersmixed with additive include but are not limited to those described inU.S. Pat. No. 7,404,971, U.S. Pat. No. 6,063,061, U.S. Pat. No.4,935,365, U.S. Pat. No. 5,015,576; U.S. Patent applications pub. No.20050287215, No. 20030064109; CultiSpher®-G and CultiSpher®-S porousgelatin microcarriers (Celltrix, Malmo, Sweden; Pereell Biolytica,Åstorp, Sweden).

In a further aspect of an embodiment, the kit contains a second syringefor containing a diluent, such as saline. Other diluents include calciumchloride diluents, and others as are known in the art. In this aspect,the diluent can be pulled from a diluent container into a syringe by theuser. Allowing users to pull diluent into a syringe allows the user tocontrol the amount of diluent used to disperse the hemostaticcomposition, and thus, control the consistency of a subsequent paste.Alternatively, the second syringe can be packaged within said kit withdiluent within the barrel. Thus, the second syringe may be pre-loadedwith diluent.

In a further aspect of the kit embodiment, the kit contains thrombin. Asnoted above, “thrombin” denotes the activated enzyme, also known asalpha-thrombin, which results from the proteolytic cleavage ofprothrombin (factor II). Thrombin can be prepared by a variety ofmethods known in the art, and the term “thrombin” is not intended toimply a particular method of production. In this aspect, the thrombinmay be provided in the kit as a lyophilized powder (see, e.g., U.S. Pat.No. 7,473,543). This lyophilized powder can be reconstituted using saiddiluent. For example, the diluent is applied from said second syringeonto said lyophilized thrombin. This can be done by adding said diluentdirectly into a vial containing lyophilized thrombin, or both thediluent and the lyophilized thrombin may be combined in a separatecontainer. The diluent may include or consist entirely of plasma. Inthis aspect, then, the kit can also contain a mixing bowl. Means ofmixing thrombin are known to those of ordinary skill in the art.

In a further aspect of the embodiment, the first syringe that containsthe hemostatic composition, and optionally an additive, and the secondsyringe that contains diluent, and, optionally, thrombin, are connectedand the content of these two syringes are passed back and forth untilthe cross-linked gelatin microspheres is fully dispersed within thediluent. The first and second syringes are connected with an adapter,wherein the adapter contains leur threads complementary to the leurthreads of the syringes. Alternatively one of syringes has a leur threadthat is complementary to the other syringe, thus the two syringes willconnect directly using complementary leur connections. This allows formixing of a hemostatic composition with diluent by connecting saidsyringes at the leur connections and passing the contents back and forthbetween the two syringe barrels by applying alternating force to theirrespective plungers until a desirable dispersion of said hemostaticcomposition is achieved. The hemostatic composition may be dry beforemixing with the diluent, though partially hydrated and even fullyhydrated hemostatic compositions can be mixed with diluent. Theresulting dispersed hemostatic compositions can be partially hydrated togreater than fully hydrated, depending on the user's preference.

One of the syringes should be sufficient to contain and dispense thedispersed hemostatic composition. Thus, the syringe needs to have asufficient barrel capacity. Following mixing of the hemostaticcomposition with diluent using said first and second syringes, thedispersed hemostatic composition are pushed to a single of the twosyringes. Large volumes of dispersed hemostatic composition will requirethat said syringe have a barrel length and width to both accommodate thedispersed hemostatic composition without making the barrel so long thata user cannot easily hold said syringe and depress its plunger with onehand. A syringe will typically contain at least 5 cc, such as at least 8cc, or at least 12 cc.

The syringe should be durable to withstand the necessary force forextruding a hemostatic composition from its barrel lumen. When thehemostatic composition is, for example, a cross-linked gelatinmicrosphere without additive, there is a sponging out effect that makesit difficult to expel the later volumes of dispersed hemostaticcomposition from a syringe. The plunger must be durable enough towithstand this force. Additive alleviates the sponging-out effect, andthus alleviates the amount of force applied to a plunger to extrude thehemostatic composition.

Thus, the hemostatic composition may be mixed with an additive. Sterilehemostatic compositions mixed with an additive include cross-linkedpolymers. Cross-linked polymers that can be mixed with an additiveinclude, but are not limited to those described in U.S. Pat. No.7,404,971, U.S. Pat. No. 6,063,061, U.S. Pat. No. 4,935,365, U.S. Pat.No. 5,015,576; U.S. Patent applications pub. No. 20050287215, No.20030064109; Cultispher®-G or Cultispher®-S macroporous gelatinmicrocarriers (Celltrix, Malmo, Sweden; Percell Biolytica, Åstorp,Sweden). The hemostatic composition mixed with additive comprises aplurality of porous, cross-linked microspheres. The cross-linked gelatinmicrospheres may be mixed with a wetting agent, and optionally apoloxamer, such as poloxamer 188, in a weight-to-weight ratio rangingfrom 60:1 to 3:1 (ratio of gelatin microsphere:poloxamer 188). Toprepare for application to a target site, the hemostatic composition iseasily and substantially homogenously dispersed in an aqueous vehicle,yielding the consistency of a fully-hydrated paste.

The additive and the hemostatic composition can be mixed and then loadedinto said first syringe as a dry powder, or partially or fully hydratedgel. Alternatively, the additive can be provided as a separate componentthat is combined with diluent (such as plasma), drawn into said secondsyringe, and then mixed with the hemostatic composition. The additiveand said hemostatic composition may be mixed as dry powders and thenloaded into said first syringe. A second syringe containing diluentalone or diluent and thrombin is then connected to said first syringeand the content of these syringes are passed back and forth between saidtwo syringes by alternating depression of their plungers. After thehemostatic composition with additive is sufficiently dispersed in thediluent, the dispersed hemostatic composition can be applied to a targetsite. The “target site” is the location to which the dispersedhemostatic composition is to be delivered. Usually, the target site isthe tissue location of interest, but in some cases the dispersedhemostatic composition may be administered to a location near thelocation of interest, e.g., when the material swells in situ to coverthe location of interest. The dispersed hemostatic composition can beextruded from said syringe directly through the orifice of said syringe.Alternatively, a suitable tip can be attached to said syringe and saiddispersed hemostatic composition can be extruded from said syringethrough the orifice of said syringe and attached tip. Tips may have alumen and orifice that is sufficient to allow passage of said dispersedhemostatic composition, but not so large that the dispersed hemostaticcomposition will drip from the orifice, or so large that the extrudedcomposition is a large, messy glob. Thus, in an aspect of the instantembodiment, the kit contains at least one tip.

The diluent in the present embodiments may also comprise, or consistentirely of, plasma, such as a patient's own plasma. In theseembodiments, for example, a patient's blood is collected and prepared bystandard procedures to obtain plasma. This autologous plasma is thenmixed with the hemostatic composition and used in the patient as thesurgeon or physician requires.

Thus, in another embodiment, there is provided composition and methodsfor delivering a dispersed hemostatic composition to a target siteneeding hemostasis. In one aspect of this embodiment, there is provideda hemostatic composition, such as a cross-linked gelatin microsphere. Inthis aspect, the cross-linked gelatin microspheres have a diameter fromabout 50 μm to about 500 μm, inclusive. In addition, the thecross-linked gelatin microspheres may further comprise pores having apore diameter of about 20 μm. Further to this aspect, the hemostaticcomposition may be mixed with a wetting agent, such as poloxamer orpoloxamer 188. Alternatively, the hemostatic composition may be mixedwith a suspending agent, such as carboxymethylcellulose. The hemostaticcomposition with or without wetting agent and/or suspending agent may beformulated into a dry powder.

In a further aspect of this embodiment there is provided a hemostaticcomposition consisting of a cross-linked polymer and an additiveselected from the group consisting of a wetting agent, a suspendingagent, and both a wetting agent and a suspending agent. The cross-linkedpolymer may be gelatin that is either dehydrothermally cross-linked,chemically cross-linked, or cross-linked by other means, such asirradiation. The cross-linked polymer can be, for example, thecross-linked polymer is a cross-linked gelatin microsphere; across-linked gelatin microsphere further comprising pores having adiameter from about 50 μm to about 500 μm, inclusive, and furthercomprising pores having a pore diameter of about 20 μm. The hemostaticcomposition is prepared into a dry powder. The additive may be a wettingagent such as poloxamer 188. Alternatively, said additive is asuspending agent, e.g., carboxymethylcellulose.

Thus, the present invention also provides for a method for delivering ahemostatic composition to a site of a body of a mammal requiringhemostasis, comprising: providing a hemostatic composition as describedherein; and applying said hemostatic composition to a site of a body ofa mammal requiring hemostasis.

The following non-limiting examples are useful in describing thecompositions and methods of the current invention.

EXAMPLES Example 1. Materials and Methods for a Making PorousCross-Linked Gelatin Microsphere

Thermal Gelation—Liquid:

Gelatin was dissolved by heating the same in water to a concentration of10% (w/v). Six (6) g of emulsifier (TWEEN® 80,polyoxyethylene(20)sorbitan monooleate) were added to 100 ml of thegelating solution. 500 ml of toluene containing 30 g emulsifier (SPAN®85, sorbitane trioleate) were then stirred into the solution. Theinitial amount of toluene was added to act as a cavity generatingcompound which is dispersed as droplets within the gelatin solution. Asmore toluene is added, the gelatin solution becomes saturated withtoluene droplets and eventually sufficient toluene is added (e.g., 500ml) so that the gelatin solution becomes aqueous gelatin dropletsdispersed in a toluene solution. When microsphere of the desired sizehad formed, the dispersion was cooled to a temperature beneath thesolidification temperature of the gelatin. This process results in theformation of gelatin microspheres which are saturated with droplets oftoluene. These toluene droplets are then removed by washing the beadswith ethanol and acetone, therewith providing a gelatin microspherewhich is filled with cavities. The gelatin beads are then cross-linkedwith glutaraldehyde, in order to further increase stability.

Thermal Gelation—Gas:

Five (5) g of emulsifier (TRITON X100™, Octoxynol-9) were added to 100ml of gelatin solution (10% w/v). Air under high pressure was then blownthrough the solution, to form a large number of air bubbles therein.Gelatin microspheres were formed by dispersing the solution in 500 mltoluene/chloroform (73/27, w/v) containing 30 g emulsifier (SPAN® 85),while stirring the system. Subsequent to obtaining microspheres of thedesired size, the dispersion was cooled, so as to solidify the gelatin.The organic solvents were then removed, by washing with ethanol andacetone. The gaseous cavity generating compound escapes automaticallyfrom the resultant gelatin microspheres due to their high porosity. Theresultant gelatin microspheres are then be cross-linked further with,for example, glutaraldehyde.

Thermal Gelation—Solid:

Ten (10) g of calcium carbonate were added to 100 ml of gelatin solution(10% w/v), thereafter, microspheres were produced in accordance withthermal gelation—gas, above. The gelatin microspheres were treated withacid, so as to dissolve the calcium carbonate and form cavities in thebeads.

Example 2. Polymerization

Acrylamid (17 g) and bisacrylamide (1.2 g) were dissolved in aTris-buffer (100 ml, 0.05 M, pH 7). Ammonium persulphate (0.5 g/ml, 0.25ml) and emulsifier (TRITON X-100™, 6 g) were added to the monomersolution. Then, 500 ml of toluene containing an emulsifier (SPAN® 85, 30g) were stirred into the system. TEMED (co-catalyst, 1.3 ml) was thenadded to the system. The organic solvents were washed out with ethanoland acetone, upon termination of the polymerization process.

Example 3. Preparation Cross-Linked Gelatin Microspheres

Gelatin was dissolved in water at a concentration of 8% (w/v) and keptat 60° C. To 100 ml solution containing TWEEN® 80 (6% w/v, Atlas Chemie,Enschede, Netherlands) toluene containing SPAN® 85 (6% w/v, AtlasChemie) was added continuously. The added toluene formed droplets in thegelatin solution until saturation with the droplet size depending on themixing speed. Through addition of excess toluene to a final volume of400 ml gelatin microspheres containing droplets of toluene wereproduced. After cooling the dispersion below 20° C., 200 ml ethanol wasadded. The formed gelatin microspheres were then further washed withethanol and after a final wash with acetone dried and overnight at roomtemperature. The dry gelatin microspheres were sieved and the fractionbetween 125 μm and 180 μm was cross-linked with glutaraldehyde (8.8%w/v) by treating for 30 min at 15° C., after reswelling in 0.1 Mphosphate buffer with pH 7.0. After removal of excess glutaraldehyde,the gelatin microspheres were heat treated at 121° C. for 20 min, whichreduced the volume to about 50%, and after washing with water andacetone finally dried overnight at 60° C.

Example 4. Gelatin Microspheres and Wetting Agent

Gelatin microspheres were combined with a wetting agent to improvehomogeneity of dispersion and syringeability. The gelatin microspherescan be prepared as described above, or purchased, e.g., Cultispher®-Smacroporous gelatin microspheres (Percell Biolytica, Åstorp, Sweden).The wetting agent is available as a dry powder, which facilitates mixingwith a dry powder microsphere. It is not necessary, however, that eitherof the powders is dry. In a one embodiment, the wetting agent is apoloxamer, such as poloxamer 188, NF (Spectrum Chemicals, Gardena,Calif., Cat. #P1169). Approximately 1 g to 60 g of Cultispher®-Smicrospheres is combined with from 1 g to 3 g of poloxamer 188, and thedry powders are mixed together until a homogenous mixture is achieved.Mixing can take place using a variety of techniques and equipment knownin the art. Alternatively, the wetting agent is a component of thediluent used to disperse the gelatin microspheres. In this embodimentthe wetting agent is present in the diluent at about 0.25% w/v to 5%w/v. For each 1 mL of diluent with wetting agent, approximately 125 mgto 175 mg of Cultispher®-S microspheres is added and then thecross-linked gelatin microspheres and the diluent with wetting agent areadmixed, for example by passing back and forth between twointerconnected syringes, until the microspheres are mixed to apaste-like consistency.

Example 5. Microsphere and Suspending Agent for Dispersion andSyringeability

Gelatin microspheres were combined with a suspending agent to improvehomogeneity of dispersion and syringeability. The gelatin microspherescan be prepared as described above. In one embodiment, the gelatinmicrospheres were Cultispher®-S microspheres (Percell Biolytica). Thesuspending agent is available as a dry powder, which facilitates mixingwith a dry powder microsphere. It is not necessary, however, that eitherof the powders are dry. In one embodiment the suspending agent is acarboxymethylcellulose, such as a medium-viscositycarboxymethylcellulose (Spectrum, Cat. #CA192). The gelatin microsphereand the suspending agent powder are mixed together until a homogenousmixture is achieved. Mixing can take place using a variety of techniquesand equipment known in the art. Alternatively, the suspending agent is acomponent of the diluent used to disperse the gelatin microspheres. Inthis embodiment the suspending agent is present in the diluent atapproximately 0.25% w/v to 5% w/v. For each 1 mL of diluent withsuspending agent, approximately 125 mg to 175 mg of Cultispher-Smicrospheres are added and then the cross-linked gelatin microspheresand the diluent with suspending agent are admixed, for example bypassing back and forth between two interconnected syringes, until themicrospheres are mixed to a paste-like consistency.

Example 6. Microspheres and Wetting Agent for Dispersion andSyringeability

Gelatin microspheres can be combined with a wetting agent and asuspending agent to improve homogeneity of dispersion andsyringeability. The gelatin microspheres can be prepared as describedabove. In a one embodiment, the gelatin microspheres are Cultispher®-Smicrospheres (Percell Biolytica). Both the suspending agent, which iscarboxymethylcellulose, and the wetting agent, which is poloxamer 188,are dry powders. The gelatin microspheres are then mixed with a powdercombination of equal parts suspending agent and wetting agent until ahomogenous mixture is achieved. Alternatively, both the suspending agentand the wetting agent are components of the diluent used to disperse thegelatin microspheres. In this embodiment, the gelatin microspheres andthe diluent with suspending/wetting agent are admixed, for example bypassing back and forth between two interconnected syringes, until themicrospheres are mixed to a paste-like consistency.

Example 7. Flowable Hemostatic Matrix

A flowable hemostatic matrix was prepared consisting of a syringecontaining the matrix coupled to another syringe containing diluent(saline, saline containing thrombin, or comparable vehicle). The matrixconsisted of cross-linked gelatin powder (Cultispher®-S macroporousgelatin microspheres) with or without additives such poloxamer 188 orcarboxymethyl cellulose. The hemostatic matrix, with or withoutadditives, was weighed and transferred into the syringe. Typically, thecompositions included 675 mg of Cultispher®-S microspheres with orwithout additives ranging from 60:1 w/w to 3:1 w/w ratio(microsphere:additive). The components were placed in a capped syringebarrel (with plunger removed) and the plunger was replaced behind thepowder. A separate syringe containing 4.5 mL of diluent was joined tothe powder syringe, using a female-to-female luer connector. The drypowder and buffer were then mixed using twenty passages. The hemostaticmatrix was allowed to hydrate for 60 sec, and the matrix was thendispensed. Dispensed preparations only containing the Cultispher®-Smicrospheres (without additive) exhibited non-uniformity regarding theaqueous content; that is, initial aliquots were more “wet” than thesubsequent aliquots. This phenomena was termed “sponging-out”. Includingwetting agents (e.g., poloxamer 188) or suspending agents(carboxymethylcellulose) as additives minimized this phenomenon,however. It was also observed and quantitatively determined using asyringe force meter that the force required to extrude the matricescontaining additives was more consistent and was minimized whendispensing the entire contents of the syringe. It was not practical todispense matrices of Cultispher®-S microspheres without additivesthrough narrow-bore administration tips that were affixed to the syringeprior to dispensing. Including wetting agents (i.e., poloxamer 188) orsuspending agents (carboxymethylcellulose) as additives minimized thisphenomenon, however, allowing the entire contents of the syringe to bedispensed with minimal force through narrow-bore administration tips.

Example 8. In Vivo Efficacy Comparison Between Non-IrradiatedCultiSpher®-S Microcarrier Beads and Solid Beads Utilizing the RatHeminephrectomy Bleed Model

A study was designed to compare hemostatic efficacy between porous,cross-linked gelatin microspheres and non-porous (solid), cross-linkedgelatin microspheres. Microspheres were prepared as above, with theexception that a single dose of 150 mg/mL thrombin was included for bothmicrosphere preparations (placebo is porous Cultispher®-S microsphereswithout thrombin).

The Mean Time to Hemostasis comparison of animals treated with theporous, cross-linked gelatin microspheres and thrombin revealed agreater reduction in TTH in animals treated versus the solidmicrospheres or placebo. Specifically, mean TTH was as follows (mean±sd)about 112±15 sec for placebo, approximately 79±37 sec for the solidmicrosphere and about 45±6 sec for porous microsphere. Without beinglimited to any particular theory, the enhanced reduction in TTH shown byporous, cross-linked gelatin microsphere group maybe explained by theporosity of the microsphere, wherein said pore creates a greater surfacearea allowing platelets to enter and mix thus creating a more rapid timeto clotting.

Another advantage of porous, cross-linked gelatin microspheres comparedto the solid beads was the consistency of the matrix. The porous,cross-linked gelatin microsphere matrix was easy to dispense and easy toapply onto the cut kidney surface from the first minute ofreconstitution until 90 min. In comparison, the solid bead matrix at 1min post-reconstitution ran off the kidney surface, and at 60 min becamevery crystalline-like and difficult to apply to the kidney surface.

Example 9. TEG Assays

Thromboelastography (TEG) assays were performed according to publishedassays. See Roche et al., 96 Anesth. Analg 58-61 (2003). A stockthrombin solution consisted of a 5000 Unit vial of recombinant thrombin(e.g., RECOTHROM®, ZymoGenetics, Inc., Seattle Wash.) dissolved in 0.5mL of 0.9% saline for a final volume of 600 μl and a concentration of8333 IU/mL. Dilutions were made of the stock in Saline plus 0.1% BSA sothat 10 μl aliquots yield a final concentration in the TEG assay between25 IU and 200 IU per ml. The 10 μl of thrombin was added to drymicrosphere powder, producing a swollen gel. This allowed a morehomogenous mixing of the thrombin and blood prior to clot initiation.Assays were performed with two TEG 5000s and monitored using commercialsoftware such as TAS 4.2.3 (HAEMOSCOPE™ Corp., Niles, Ill.).

Blood samples were obtained from rabbits prior to and after treatmentwith clopidogrel bisulfate. Each animal received three, daily 20 mg/kgoral doses of clopidogrel bisulfate. A 75 mg clopidogrel bisulfatetablet was crushed and suspended in 3 mL of sterile distilled water. Therabbits received between 1.6 to 2.2 mL of the suspension by gavage.Blood samples were taken after the third daily treatment. According toTEG protocols, on Day 1 of the AV shunt model, the rabbit blood is named“Rabbit Mo/Day”. This name is the same for each subsequent collection ofthe same rabbit's blood through out the three-day experiment. Forexample, on Day 1 pretreatment blood and blood 2 hr after theclopidogrel bisulfate gavage is received on November 12 and named “Rb11/12.” On Day 2, 11/13 and Day 3, 11/14 the sample remains Rb 11/12.Blood was collected in citrate unless specified. Blood was re-calcifiedin the TEG assay cup with 20 μl of 0.2M CaCl₂, 0.9% NaCl, pH 7.4 (stockCaCl₂) as described in the HAEMOSCOPE™ TEG protocol.

The data in FIG. 1 was generated using protocol 2 for microspheregel-only and protocol 3 for microsphere gel and thrombin. The assays inFIGS. 2, 3, and 4 were performed using protocol 5 with normal citratedblood with or without heparin. Heparin was added as described inprotocol 4.

Protocol 1. Minus-Heparin Assay:

Prior to the addition of heparin, the blood was recalcified to determinethe pre-heparin TEG parameters. The results of the heparin free assaysare similar to for both the pretreatment and clopidogrel bisulfatetreated blood samples. Some of the clopidogrel bisulfate blood sampleshave an early R value suggesting that they are hyper-coagulable. Theseresults are consistent with those reported in the art.

Protocol 2. Minus-Heparin Assays with Microsphere Gel Present:

The mixing of microsphere gel may be modified while developing theparticular method. For example, one microsphere gel preparation was madeby allowing gel to swell fully, and then adjusted to a 1:1 suspension in0.9% NaCl. The gel was pipetted into the TEG cup just prior to theassay.

Protocol 3. Thrombin Plus Microsphere Gel:

Dry microsphere powder (23.5 g) was mixed with 4.7 mL of 5000 IU/mLrecombinant thrombin and 56.9 mL of 0.9% Saline. This resulted in ahydrated gel with 1 IU thrombin per mg dry microsphere powder. The samegel to saline ratio (1:2.6) was used as a minus thrombin control.Various amounts of the hydrated gel was weighed into a TEG assay cupusing a spatula just prior to the assay.

Protocol 4. TEG Assays with Heparin:

Heparin was added to the blood (1 IU heparin/mL blood) in order todetermine the effect of heparin alone and heparin plus clopidogrelbisulfate on the reaction time (R), reaction rate (K) and maximumamplitude (MA) for all three days of clopidogrel bisulfate treatment.The last blood sample on Day 3 is assayed with heparin transfused intothe rabbit (heparin ‘on board’) just prior to the start of the AV shuntmodel. The transfused heparin is approximately 1 unit/mL blood. Thislevel of heparin is just sufficient to obtain a APTT of >400 seconds.

Protocol 5. Thrombin Activation of Heparinized Blood:

Prior to the assay, a suspension of either 50 mg or 100 mg ofmicrosphere gelatin beads in 1.5 mL of distilled USP irrigation waterwas allowed to swell for 15 min by rocking. Then, 35 μl of the gelatinbead suspension was dispensed into each assay cup and allowed to dry at37° C. for 30 min. The dry gel is either 1.21 mg or 2.42 mg per cuprespectively.

The TEG assay is composed of 330 μl Blood, 20 μl stock CaCl₂ solution,10 μl thrombin ranging in concentration between 25 U and 8333 U inmicrosphere gel for a total volume of 360 μl.

When high thrombin is added directly into blood by standard pipettingtechniques the fibrinogen is converted to fibrin faster than mixing canoccur. This results in a non-homogenous clot. Observational dataconfirms this: (a) the TEG maximum amplitude (MA) may collapse andstabilize at a lower value; and (b) the pipette tip may suck in part ofthe clot during mixing.

In order to overcome problems mixing, microsphere gel was added to theTEG assay cup. The swelling of the microsphere gel occurs completelyduring the first minute. FIG. 4A shows the reaction rate of the clot(R). R is the minutes from the start of the assay to the firstmeasurable clot strength (2 mm amplitude). When the thrombin is added todry microsphere gel, the R is increased indicating that the clotkinetics is slower than when thrombin is added directly. The predictionthat thrombin entrapment within microsphere pores would allow bettermixing was also supported by the maximum clot strength MA (FIG. 4B). MAis related to G by the formula: G=5000 MA/(100−MA). Without themicrosphere gel, the MA actually declined with increasing thrombin,whereas the thrombin plus microspheres gel maintained strength. Thediffusion rate of thrombin from the gel was inferred to be slowerbecause the reaction rate was slower.

The microsphere gel served as a pro-coagulant as well as a matrix forthe delivery and mixing of thrombin. This is may be due to theactivation of the Contact Pathway by cross-linked gelatin. Thecross-linked gelatin may also activate platelets via receptors such asGP VI, the collagen receptor.

Clot formation in the presence of heparin first requires the saturationof Anti-thrombin (primarily AT III). Once anti-thrombin is overcome,fibrin formation and platelet activation via the thrombin receptors canoccur. Thrombin activation of platelets also bypasses plateletactivation inhibitors such as clopidogrel bisulfate and aspirin. Onewould predict that such thrombin activation would not bypass plateletaggregation inhibitors INTEGRILIN® (eptifibatide, Schering-Plough,Kenilworth, N.J.), REOPRO® (Abciximab, Centocor B.V., Leiden,Netherlands), and AGGRESTAT® (tirofiban, Merck & Co, Inc, WhitehouseStation, N.J.).

Example 10. Clotting in the Presence of Microsphere/ThrombinFormulations

Blood was collected in citrate and calcium (11 mM) was added back toinitiate the assay. Under these conditions there is sufficient thrombingenerated from prothrombin in the blood to form a strong clot. Withoutcoagulation inhibitors in the blood (‘on board’) the primary advantagein adding thrombin to stanch bleeding is to decrease the clotting timefrom 12-21 min to less than 1 min. This can be accomplished with about 3IU thrombin/mL of blood (FIG. 1, microspheres and thrombin).

Microsphere gel was also found to strengthen clots. The uninhibitedcoagulation system in normal blood generates a strong clot without addedthrombin. Clot strength may be increase, however, by adding increasingamounts of microsphere gel to the blood. Clots formed with microspheregel and thrombin exhibit a >40% increase in strength (FIG. 1). Theseclots were formed with relatively low thrombin concentrations ≤3 IU/mL.

High thrombin concentration plus microsphere gel is advantageous whencoagulation inhibitors are present in the blood. Under these conditionsvery large amounts of thrombin are typically required to overcomeclotting inhibition. As shown in FIG. 2 (strength) and FIG. 3 (reactiontime), in which clots assayed with normal citrated blood and clots ofblood inhibited with 1 IU/mL heparin are superimposed. There is a100-fold increase in the thrombin concentration required to overcomeheparin inhibition. The actual concentration of thrombin in themicrosphere gel (93 μl gel, fully swollen, per ml blood) is aboutten-times greater than the amount in the blood. The thrombinconcentration required in the gel to form a clot is thereforeapproximately 750 IU to 2000 IU thrombin/mL gel. This clot does notobtain the strength of the heparin-free blood, but is within the rangeof clot strength considered normal for human blood (Roche et al., 96Anesth. Analg 58-61 (2003)). The addition of clopidogrel bisulfate toheparin increases the requirement for thrombin even more (FIG. 6).Clopidogrel bisulfate inhibition is usually detected by platelet mappingbut this data suggests that it may also be measured in the presence ofheparin when exogenous thrombin is added.

Example 11. In Vivo Testing of Hemostatic Compositions ComprisingThrombin

A rabbit vascular anastomotic bleeding model was employed. PTFE arterialvenous grafts were punctured four times with a 4-0 suture needle,immediately wrapped with absorbable gelatin sponge, USP, soaked withplacebo (vehicle) or rThrombin (1000 IU/mL or 125 IU/mL). Overall meanTTH was calculated for animals treated with placebo, standardized dosesof heparin alone or heparin with clopidogrel bisulfate. Results wereanalyzed using linear models with robust standard error estimates.

rThrombin at a concentration of 1000 IU/mL completely reversed theeffects of heparin or heparin with clopidogrel bisulfate on TTH, whereasrThrombin 125 IU/mL did not. rThrombin 1000 IU/mL resulted in asignificantly lower mean TTH compared to 125 IU/mL (p<0.0001) in bothheparin or heparin with clopidogrel bisulfate-treated animals, and bothconcentrations of rThrombin were superior to their respective placebocontrols (p<0.001).

Potent platelet inhibition is commonly encountered in vascular andgeneral surgery; over 18 million prescriptions for clopidogrel bisulfateare written annually in the U.S. In this in vivo study, rThrombin 1000IU/mL resulted in a significantly lower mean TTH than 125 IU/mL andcompletely reversed the effects of heparin or heparin with clopidogrelbisulfate. These results support the broad clinical utility of topicalrThrombin at 1000 IU/mL as the standard of care.

Similarly, when such different thrombin concentrations are applied inthe context of the hemostatic microspheres of the present invention, asimilar effect is expected; that higher thrombin concentrations of 1000IU/mL or more have a lower mean TTH. Moreover, the in vivo data supportsthe potential application of higher thrombin concentrations in thehemostatic microspheres in applications where such higher concentrationsare desirable.

Example 12. In Vivo Testing of Hemostatic Compositions ComprisingThrombin

In Vivo Study Design: The various treatment groups are shown in Table 1for different rThrombin concentrations, and Table 2 for animalspretreated with clopidogrel bisulfate.

For each set of experiments, control parameters were used to demonstrateconsistency among animals and standardization between grafts include:body weight, body temperature baseline/terminal for each graft,baseline/terminal mean arterial pressure (MAP) for each graft,baseline/terminal AV shunt blood flow rate for each graft, and baselineand post heparin treatment activated partial thromboplastin time (APTT).

TABLE 1 Study Design with Varying Concentrations of rThrombin TreatmentGroups Units of rThrombin (IU/mL) No. Grafts/Group AGS + Placebo 0 12AGS + rThrombin 31.25 8 AGS + rThrombin 62.5 8 AGS + rThrombin 125 8AGS + rThrombin 1000 8 AGS = absorbable gelatin sponge, USP, TTH = Timeto hemostasis

TABLE 2 Study Design with 125 IU/mL or 1000 IU/mL in Rabbits Pretreatedwith clopidogrel bisulfate and Heparin No. Treatment Groups^(a)Anticoagulant Grafts/Group AGS + Placebo clopidogrel bisulfate/heparin10 AGS + rThrombin clopidogrel bisulfate/heparin 14 125 IU/mL AGS +rThrombin clopidogrel bisulfate/heparin 14 1000 IU/mL AGS = absorbablegelatin sponge, USP, TTH = Time to hemostasis ^(a)Data not shown-rabbitstreated with clopidogrel bisulfate alone but no heparin for controlpurposes

Animals:

Approximately 12-week-old female New Zealand White rabbits, weighing 2.0kg to 3.8 kg, Lot No. 2525 (Charles River Laboratories, HollisterCalif.) were used for this study. Animals were acclimated to thefacility for 7 days to 10 days before the experiment, and weremaintained in good condition at the ZymoGenetics Vivarium. The studyprotocol was approved by the Institutional Animal Care and UseCommittee.

Absorbable Gelatin Sponge and Test Articles:

A gelatin sponge (GELFOAM® absorbable gelatin compressed sponge, USP,Pharmacia & Upjohn Co., Kalamazoo, Mich., size 100) was cut into 2×4×1cm strips and combined with test article (rThrombin or placebo).

Recombinant human thrombin, rThrombin, (RECOTHROM®, ZymoGenetics, Inc.,Seattle, Wash.) material consisted of a 5,000 IU/vial of lyophilizedproduct manufactured by Abbott Laboratories. On the day of the study, avial of rThrombin was removed from the refrigerator, and allowed to sitat room temperature for a minimum of 20 to 30 min before beingreconstituted in 5 mL of sterile saline yielding a 1000 IU/mL solution.The 1000 IU/mL solution was further diluted with vehicle to yield 31.25,62.5, and 125 IU/mL solution. Vehicle solution consisted of theformulation for rThrombin without active ingredient: 1.6 mM Histidine.200 mM NaCl, 1.28 mM CaCl₂, 0.96% w/v sucrose, 1.28% w/v mannitol,0.032% PEG-3350, and pH adjusted to 6.0.

Clopidogrel Bisulfate Administration:

Each animal received three daily 20 mg/kg oral doses of clopidogrelbisulfate prior to undergoing AV shunt. A 75 mg clopidogrel bisulfatetablet was dissolved in 3 mL of sterile water yielding a workingconcentration of 25 mg/mL. Each animal received between 1.6 2.2 mL ofthe 25 mg/mL solution per day by gavage.

Rabbit AV Shunt Procedure:

Each rabbit was weighed and immobilized with Ketamine hydrochloride (50mg/kg) via intramuscular injection. The animal was placed on thesurgical table in a nose cone connected to a precision gas anesthesiavaporizer, which delivered anesthesia (ISOFLO® isoflurane, USP, AbbottLabs., North Chicago, Ill.) vapor concentration of 4% to 5% forinduction, and 1% to 2% for maintenance of a surgical plane ofanesthesia, with a flow rate of 1% to 2% L/min O₂. The animal was placedon a water-jacketed heating pad maintained at 37° C. during theexperimental period, and a rectal thermoprobe was inserted formonitoring of body temperature. Blood pressure, mean arterial pressure,and body temperature were measured throughout the experiment.

To create the AV shunt, a skin incision was made on ventral surface ofthe neck. The right external jugular vein and left common carotid arterywere isolated and cannulated with 3 to 4 cm length Micro-Renathanetubing (MRE 080, 0.080 O.D.″×040 I.D.″, Braintree Scientific, Braintree,Mass.) which was connected to a 15 cm length of silicone catheter tubing(7-French, 0.078 LD″×0.125 O.D.″, Access Techs., Skokie, Ill.). Thecatheters were exteriorized and connected with a 3 mm diameter, 2 cm to2.5 cm long polytetrafluoroethylene (PTFE) graft segment (BardPeripheral Vascular Inc., SN AFEP 7108) producing an arterial-venousshunt linking the blood flow of the left carotid artery and the rightjugular vein. Blood flow through the shunt was measured using aTransonic System Inc. Flowmeter model TS410 (Ithaca, N.Y.).

Each rabbit received 100 U/kg intravenous (i.v.) bolus injection ofheparin followed by a continuous 50 U/kg/mL infusion of heparin (porcinederived, Abraxis Pharma. Prods., Schaumburg, Ill.) at a flow rate of 5mL/hr via the femoral vein. Prior to heparin treatment and approximately5 min to 10 min post i.v. heparin bolus treatment APTT was measured. Inorder to assure that the animal was anticoagulated (APTT >400 seconds)the animal received a 50 U/kg i.v. bolus injection of heparin everythird graft.

Suture Hole Bleed and Measurement of TTH:

Prior to creating suture holes in any of the grafts, a baseline MAP ofapproximately 55 mm Hg had to be achieved and an APTT value greater than400 sec. Assessment of suture hole bleeding consisted of puncturing thecenter section of the PTFE graft segment with a 2 4-0 18 inch silksuture needle (reverse cutting needle size P-3, Ethicon Inc.,Somerville, N.J.) creating four needle holes.

Immediately following suture hole punctures the absorbable gelatinsponge (AGS) containing test article was wrapped around the graftcompletely covering the suture holes. The AGS containing test articlewas immediately covered with gauze sponges and continuous digitalpressure applied for 60 sec of the 5-min study period. At the end of the60-sec period, the gauze sponges were visually inspected for bleeding.If cessation of bleeding did not occur, the gauze sponges were replacedwith new gauze sponges, test article was reapplied followed by digitalpressure. This process was repeated until no visible blood was observedon the gauze sponges. Time to hemostasis was recorded in seconds. In theevent TTH was not achieved within 300 sec the study was terminated and300 sec was recorded. At the conclusion of the 5-min study period, theclot at suture hole sites was assessed is some of the grafts. Followingassessment of clot burst the catheters were clamped flushed with saline,the polytetrafluoroethylene (PTFE) segment was removed and fresh PTFEsegment was inserted. Once blood pressure achieved a reading of 55 mm Hgthe process was repeated. In the event 55 mm Hg MAP could not beachieved, the animal was euthanized.

Suture Hole Clot Burst Assessment:

In some of the heparin-only treated rabbits, and in all of theclopidogrel bisulfate/heparin treated rabbits, clot burst at the suturehole sites was assessed at the end of the 5 min experimental, providedTTH was achieved. The procedure involved clamping off blood flow to thejugular vein catheter approximately 2 cm to 3 cm downstream from thegraft for a period of 10 sec. Complete obstruction of blood flow createdan increase in blood pressure from the arterial flow. In the event bloodseeped through the AGS during the 10-sec-period then clot burst wasrecorded as being positive. IF no leakage was observed the clot burstwas recorded as being negative.

Statistical Analysis of TTH:

The linear model for time to hemostasis is given by where Y is TTH inseconds. X is rThrombin dose group as a categorical variable, Z is theclopidogrel bisulfate treatment coded as “0” for no clopidogrelbisulfate and “1” for with clopidogrel bisulfate, and is the interactionbetween rThrombin dose group and clopidogrel bisulfate treatment.

Results:

The primary efficacy endpoint for this study was time to hemostasis(TTH). The TTH values ranged from 65 sec to 300 sec, and mean TTH wascalculated for each treatment group (Table 3, below). Comparison of thefour concentrations of rThrombin versus vehicle/control demonstrated aconcentration dependent reduction in TTH. A significant reduction in TTH(p<0.001) was observed between grafts treated with 62.5 IU/mL, 125 IU/mland 1000 IU/mL rThrombin+AGS (142±35.7, 87±22.5, 71±4.6) as compared toplacebo plus AGS (249±67.1) and 31.25 IU/mL rThrombin plus AGS(213±39.3).

TABLE 3 Group Mean ± SE TTH for Each Group, Following Treatment withrThrombin at Increasing Doses rThrombin Number of Treatment Groups DoseIU/mL Grafts/Group TTH ± SE (s) AGS + Vehicle 0 12 249 ± 67.1 AGS +rThrombin 31.25 8 213 ± 39.3 AGS + rThrombin 62.5 8 142 ± 35.7 AGS +rThrombin 125 8  87 ± 22.5 AGS + rThrombin 1000 8 71 ± 4.6 AGS =absorbable gelatin sponge, USP

Even in the presence of a systemic anti-coagulant like clopidogrelbisulfate, rThrombin significantly reduced the TTH in the rabbit AVshunt model (Table 4). A significant reduction in TTH was observed ingrafts treated rThrombin (both 125 IU/mL and 1000 IU/mL) plus AGS, ascompared to vehicle plus AGS (p<0.001). The reduction in mean TTHobserved in grafts treated with 1000 IU/mL rThrombin was significantlygreater, however, as compared to the 125 IU/mL rThrombin group(p<0.0001). It should also be noted that the variability of the TTHmeasurement was much less with in grafts treated with the higherconcentration of rThrombin. Thus, the concentration of rThrombin at thewound site had a highly significant effect on TTH in the model in thepresence of the anticoagulant clopidogrel bisulfate.

TABLE 4 Group mean ± SE TTH for Each Group, Following clopidogrelbisulfate Treatment with or without rThrombin Number of Treatment GroupsAnticoagulant Grafts/Group TTH ± SE(s) AGS + Placebo clopidogrelbisulfate/ 10 270 ± 32.5 Heparin AGS + rThrombin clopidogrel bisulfate/14 183 ± 62.2 125 IU/mL Heparin AGS + rThrombin clopidogrel bisulfate/14 73 ± 8.4 1000 IU/mL Heparin AGS = absorbable gelatin sponge, USP

Model control parameters included: body weight, body temperaturebaseline/terminal for each graft, baseline/terminal mean arterialpressure (MAP) for each graft, baseline/terminal AV shunt blood flowrate for each graft, and baseline post heparin treatment activatedpartial thromboplastin time (APTT). Individual animal body weight,starting and ending study APTT were similar between groups. Comparisonof baseline APTT and terminal APTT showed a 2.6-fold or greater increasevalues in terminal APTT, which was expected due to heparinadministration. Group mean comparison of body temperature, MAP, bloodflow and amount of test article applied to each gelfoam were similar foreach group.

In FIG. 5, analysis of the mean TTH demonstrates significant reductionin TTH in grafts treated with either dose rThrombin as compared to itsrespective placebo control (p<0.001). Also, a significant reduction ingroup mean TTH was observed in grafts treated with 1,000 IU/mL rThrombinas compared to the 125 IU/mL rThrombin group (p<0.0001). A secondaryendpoint of interest was an evaluation of the strength of clots formedunder the various conditions of rThrombin concentration andanticoagulation. Clot burst at the suture hole sites was assessed at theend of the 5 min experimental, provided TTH was achieved. No significantdifferences in incidence of clot rupture were observed between thedifferent rThrombin concentrations tested in rabbits anti-coagulatedwith heparin alone. In the clopidogrel bisulfate treated rabbits, therewas a much higher incidence of clot rupture, however, in grafts treatedwith 125 IU/mL of rThrombin (79%), as compared with 0% of the graftstreated with 1,000 IU/mL of rThrombin (FIG. 6).

The photos show the rabbit AV shunt model grafts used in clot burstassessment with either 125 IU/mL rThrombin (top three photos), or 1,000IU/mL rThrombin. It was observed that 79% grafts treated with 125 IU/mLtested positive for clot burst at the suture hole site as compared to 0%of the grafts treated with 1,000 IU/mL of rThrombin.

Similarly, when such different thrombin concentrations are applied inthe context of the hemostatic microspheres of the present invention, asimilar effect is expected; that higher thrombin concentrations of 1,000IU/mL or more have a lower mean TTH. Moreover, the in vivo data supportsthe potential application of higher thrombin concentrations in thehemostatic microspheres in applications where such higher concentrationsare desirable.

Example 13. In Vitro Study

TEG Assay Preparation; Samples from Rabbit AV Model:

Blood was collected 2 hr post-clopidogrel bisulfate treatment asdetailed in the AV shunt model methods. The blood was collected incitrate and re-calcified in 20 μl of 0.2 M CaCl₂, 0.9% NaCl, pH 7.4,just prior to the assay according to the TEG assay protocol. One unit/mlheparin was added to in vitro to samples from rabbits duringpre-clopidogrel bisulfate treatment and for Day 1 samples. The samplesfrom the rabbits after Day 3 clopidogrel bisulfate treatment includedheparin that was transfused into rabbits such that the heparin isapproximately 1 U/mL of blood (animals had an APTT of >400 seconds).

Adaptations to TEG Method to Allow Analysis of Exogenous rThrombinAddition:

The citrated rabbit blood was recalcified according to the standard TEGprotocol. See Roche et al., 96 Anesth. Analg 58-61 (2003). The bloodclotted within normal parameters for rabbit blood without exogenousthrombin. Direct addition of 0.76 to 6.1 IU/mL rThrombin initiates clotformation at a rate that precludes TEG measurement. A modified method ofthrombin addition Haemoscope covering a ten-fold range in the amount ofthrombin was used for clot initiation.

Thromboelastograph Study Design:

TEG measures time latency for initiation of the clot, time to initiationof a fixed clot firmness of 20-mm amplitude,

-   -   kinetics of clot development as measured by the angle (α)    -   maximum amplitude (MA) of the clot    -   R value (reaction time) is measured from the beginning of the        tracing to the point where the curve is 1 mm wide    -   Clot strength (shear elastic modulus) is defined as G        (dynes/cm²).

Results:

The in vivo rabbit experiments (Example 11 and Example 12) indicatedthat increasing doses of rThrombin could decrease TTH in the AV shuntmodel. FIG. 6 plots clot strength G (Dynes/cm²) for clots from rabbitsprepared for the AV shunt model. Blood was collected from rabbits twohours after administration of clopidogrel bisulfate. The blood wascollected in citrate recalcified, and used in the TEG assay. Samplesfrom pretreatment and Day 1 elopidogrel bisulfate treated rabbits wereincubated with 1 U/mL heparin in vitro, and heparin added during the AVshunt procedure for the Day 3 clopidogrel bisulfate sample.

In experiments conducted in rabbits not pretreated with clopidogrelbisulfate, increasing the concentration of rThrombin used in vitroincreased the clot strength (shear elastic modulus) as defined as G(dynes/cm²), shown on the Y-axis in FIG. 6A, and demonstrated an EC50 of54 U/ml. In further experiments, clot strength for samples from rabbitspretreated with clopidogrel bisulfate for 2 hr can be seen in FIG. 3B,with an EC50 of 66 U/mL. In both cases, the strength of clot increasedwith increasing rThrombin concentrations, supporting the conclusionsfrom the in vivo AV shunt model.

Similarly, when such different thrombin concentrations are applied inthe context of the hemostatic microspheres of the present invention, asimilar effect is expected; that higher thrombin concentrations of 1,000IU/mL or more have a lower mean TTH. Moreover, the in vitro datasupports the potential application of higher thrombin concentrations inthe hemostatic microspheres in applications where such higherconcentrations are desirable.

Example 14. Confirmation of Hemostatic Activity in Gelatin MicrosphereMatrix

To confirm that the hemostatic activity of recombinant human thrombin(rThrombin) is maintained when applied using the gelatin matrix and toevaluate performance characteristics of the matrix, non-GLP pharmacologystudies were conducted using previously established bleeding models, ratheminephrectomy and rabbit liver injury. An additional modelrepresenting a type of bleeding common in vascular surgery procedureswas also used to evaluate the rThrombin gelatin matrix. Key aspects ofthis model, which produced bleeding from a needle puncture in anarterial graft site, include a higher pressure and flow rate present atthe bleeding site, and the use of anticoagulant in the animals.Together, these three bleeding models were used to confirm theperformance and hemostatic activity of the rThrombin gelatin matrixunder a range of conditions mimicking the intended clinical use.

Example 14A: In Vivo Study: Rat Kidney Bleeding

rThrombin and placebo, both applied with gelatin matrix, were comparedin a blinded manner in the rat heminephrectomy model (n=8 per group).Briefly, this model involves creation of a standardized injury to thekidney, using a template to produce a sagittal cut and removeapproximately 18% of the kidney mass. Test article (gelatin matrixsuspended in solution containing rThrombin) or placebo (gelatin matrixsuspended in solution containing the formulation for rThrombin withoutactive ingredient) was applied to the cut surface via syringe, two gauzesponges were placed over the test article, and continuous digitalpressure was applied for 30 sec. At the end of the 30 sec period, thegauze sponges were visually inspected for bleeding. In the event thattime to hemostasis (TTH) was not achieved, gauze sponges were replacedand additional test article was applied. Digital pressure was alternatedwith visual inspection every 10-15 sec until TTH was achieved, up to amaximum time of 10 min. Time to hemostasis was noted when no visibleblood was seen soaking through to the clean gauze sponges. Mean arterialblood pressure and body temperature were monitored throughout theexperimental period. Time to hemostasis is shown in FIG. 7A orindividual animals treated with rThrombin or placebo. A significantreduction in group mean TTH (p<0.0001, t-test) was observed in woundstreated with rThrombin applied using gelatin matrix as compared toplacebo applied in the same manner. Hemostasis was eventually achievedwith the placebo/gelatin treatment. The mean TTH was approximately3-fold greater in this group than in the rThrombin/gelatin group,however. These data demonstrate the importance of rThrombin as theactive hemostatic agent in this model.

Example 14B: In Vivo Study: Rabbit Liver Bleeding

rThrombin and placebo, both applied with gelatin matrix, were comparedin a blinded manner in the rabbit liver injury model (n=6 per group).Briefly, this model involves creation of a standardized injury to thesurface of the left medial liver lobe, using a template to remove asection of approximately 2 cm diameter. Test article (gelatin matrixsuspended in solution containing rThrombin) or placebo (gelatin matrixsuspended in solution containing the formulation for rThrombin withoutactive ingredient) was applied to the cut surface via syringe, followedby placement of two gauze sponges over the test article and applicationcontinuous digital pressure for 60 sec. At the end of the 60-sec-period,the gauze sponges were visually inspected for bleeding. In the eventthat TTH was not achieved, gauze sponges were replaced and additionaltest article was applied. Digital pressure was alternated with visualinspection every 10-15 sec until TTH was achieved, up to a maximum timeof 10 min. Time to hemostasis was noted when no visible blood was seensoaking through to the clean gauze sponges. Mean arterial blood pressureand body temperature were monitored throughout the experimental period.Time to hemostasis is shown in FIG. 7B for individual animals treatedwith rThrombin or placebo. A significant reduction in group mean TTH(p=0.0016, t-test) was observed in wounds treated with rThrombin appliedusing gelatin matrix as compared to placebo applied in the same manner.Although hemostasis was eventually achieved with the placebo/gelatintreatment, the mean TTH was more than two-fold greater in this grouprelative to that in the rThrombin/gelatin group. These data demonstratethe importance of rThrombin as the active hemostatic agent in thismodel.

Example 14C: In Vivo Study: Rabbit AV Shunt

A rabbit A-V shunt model was developed to mimic bleeding that couldoccur in a vascular surgery setting. Briefly, this model involvesproducing an arterial-venous shunt linking the blood flow of the leftcarotid artery and the right jugular vein, using a PTFE graft segmentabout 2 cm in length to connect the catheters. Rabbits were treated withintravenous Heparin (100 U/kg i.v. bolus and 50 U/kg/hr) to maintainshunt patency. Mean arterial blood pressure (MAP), blood flow rate inthe shunt, APTT, and body temperature were monitored throughout theexperimental period. A baseline MAP of approximately 55 mm Hg wasachieved prior to puncturing any of the grafts. Immediately followinggraft puncture with a suture needle, (reverse cutting needle size P-3),the test article or placebo was administered using a syringe for thegelatin matrix groups or a spray pump for liquid rThrombin groups.Grafts were immediately covered with gauze sponges and continuousdigital pressure was applied for 60 sec. In the grafts treated withspray application placebo, the treatment was essentially equivalent toapplication of direct pressure alone. At the end of the 60 sec period,the gauze sponges were visually inspected for bleeding. In the eventthat TTH was not achieved, gauze sponges were replaced and additionaltest article was applied. Digital pressure was alternated with visualinspection every 10-15 sec until TTH was achieved. Time to hemostasiswas noted when no visible blood was seen soaking through to the cleangauze sponges. In the event TTH was not achieved within the 5-minutestudy period, the study was terminated and 300 sec was recorded. At theconclusion of the study period, the catheters were clamped, flushed withsaline, and a new PTFE segment was installed, provided the 55 mm Hg MAPcriteria was met. Test articles were randomized among twenty-four graftsin four different animals for this study. A generalized estimatingequations model was used to compare TTH measurements between treatmentgroups using SAS (Version 9.1.3).

The primary purpose of the rabbit AV shunt model study was to confirmthe hemostatic activity of rThrombin and evaluate performance of thegelatin matrix in a vascular bleeding application. rThrombin andplacebo, both applied with gelatin matrix, were compared in a blindedmanner in the rabbit A-V shunt model. Time to hemostasis (TTH) is shownin FIG. 7C for individual graft sites (n=6/group) treated with rThrombinor placebo applied using the gelatin matrix. A significant reduction ingroup mean TTH (p<0.0001) was observed in grafts treated with rThrombinas compared to placebo. All measurements of TTH for therThrombin-treated grafts had the lowest possible value of 60 sec,compared to a much higher mean TTH in placebo-treated grafts includingtwp grafts in which hemostasis was not observed (300-sec maximal value).

A secondary purpose of this study was to assess the relativecontributions of rThrombin and the gelatin matrix to hemostaticactivity. Data from the rat heminephrectomy and rabbit liver injurymodel suggested that the matrix itself may possess some limitedhemostatic activity independent of rThrombin. A passive hemostaticeffect of the gelatin matrix is expected, based on its physicalproperties and ability to slow the flow of blood from a wound. Underconditions of the AV shunt model, however, the larger and moreconsistent difference between treatment groups clearly indicate thatrThrombin provides the primary mode of action. This conclusion isfurther supported by comparing the effects of rThrombin delivered in theabsence of gelatin matrix. This was accomplished by spray application ofrThrombin, with blinded comparison to placebo. A significant reductionin group mean TTH was observed in grafts treated with rThrombin alone(FIG. 7C) as compared to the gelatin matrix/placebo group (p<0.0001), orto the group treated with placebo alone (p<0.0001).

Example 15. Comparison of Microsphere Size in rThrombin-Assisted TTH

Gelatin microspheres may be sieved to obtain microparticles of aparticular size range. In the case of CultiSpher®-S macroporous gelatinmicrocarrier microspheres, the material can be sieved to include sizesof about 130 μm to 380 μm diameter. During production of a batch,approximately half of the material may be lost as the fraction ofmicrospheres of about <130 μm diameter. A comparison of flowablethrombin devices including all gelatin microsphere material with a sizeof <380 μm diameter with a flowable device including gelatin microspherematerial with a size of about 130-380 μm diameter was undertaken.

More specifically, rThromin in CultiSpher®-S macroporous gelatinmicrocarrier N18051, size about 130-380 μm diameter, was compared withrThrombin in CultiSpher®-S macroporous gelatin microcarrier 19122, sizeabout <130 μm diameter mixed at a 50:50 weight ratio with microcarriersof 130-380 μm diameter. When tested in the rat heminephrectomey model, ahigher and more variable TTH was observed in the mixed batch having50%<130 μm diameter than in the batch having standard 130-380 μmdiameter formulation (FIG. 8). Additionally, greater adherence to gauzeand re-bleeding was observed in the mixed batch of 50%<130 μm diameterthan the standard batch of 130-380 μm diameter formulation. Includingsmaller microparticles lead to reduction in efficacy in this model, andbeads with the size range of about 130 μm to about 380 μm in diameterproved more effective.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. A kit including a hemostatic compositioncomprising: a plurality of porous cross-linked gelatin microspheres; awetting agent; a suspending agent; and thrombin wherein the wettingagent and the suspending agent are present in a ratio ofmicrospheres:the wetting agent and the suspending agent of 3:1 to 60:1(w/w), wherein the wetting agent is poloxamer 188 and the suspendingagent is carboxymethylcellulose, wherein the porous cross-linked gelatinmicrospheres have a diameter from about 50 μm to about 500 μm,inclusive, when fully hydrated, and wherein the porous cross-linkedgelatin microspheres have a pore diameter from about 15 μm to about 25μm, inclusive.
 2. The kit of claim 1, further comprising a hemostaticcomposition delivery device comprising at least one syringe.
 3. The kitof claim 2, wherein the syringe contains the hemostatic composition. 4.The kit of claim 1, further comprising a diluent.
 5. The kit of claim 3,wherein the delivery device further comprises a second syringe and adiluent, wherein said diluent, optionally, contains or is plasma.
 6. Thekit of claim 5, wherein the thrombin is present in the diluent.
 7. Thekit of claim 5, wherein the second syringe and the syringe containingthe hemostatic composition are interconnected.
 8. The kit of claim 7,wherein the syringes are interconnected by a male/female luer locksystem.
 9. The kit of claim 1, wherein the hemostatic composition is adry powder.
 10. The kit of claim 1, wherein the thrombin concentrationis in a range of about 1,000 IU to about 2,000 IU per mL of rehydratedmicrosphere gel, inclusive.
 11. The kit of claim 1, wherein the thrombinconcentration is in a range of about 1,000 IU to about 5,000 IU per mLof rehydrated microsphere gel, inclusive.
 12. The kit of claim 1,wherein the thrombin concentration is in a range of about 5,000 IU toabout 50,000 IU per mL of rehydrated microsphere gel, inclusive.
 13. Thekit of claim 1, wherein the hemostatic composition has been compressedinto a pledget and dried.